The broad, long-term objective of this research program is to define at the molecular level the mechanism(s) and regulation of signal transduction by the two receptors for thyrotropin-releasing hormone (TRH-R1 and TRH-2). It is hoped that these studies will lead to a better understanding of cell regulation under physiologic and pathologic circumstances by all hormones, growth factors and neurotransmitters that signal via G protein-coupled receptors. The findings may lead to rational design of new drugs to treat diseases of these signaling systems. This is timely because new insights into these diseases are now being gained and a combined approach using molecular biology and computer modeling holds great promise for a deeper understanding of these processes. Three interrelated aspects of the biology of TRHs will be explored. Specific aims are: 1) To determine the molecular details of TRH binding and the conformational changes in the transmembrane helices of TRH-R1 that mediate activation; 2) To delineate the differences in the biology of TRH-R1 and TRH-R2 and determine whether these differences may be mediated by differences in their carboxyl termini, and 3) To identify cellular proteins with which the carboxyl termini of TRH-R1 and R2 may interact so as to understand the molecular mechanism(s) of TRH-R function and differences in signaling of TRH-R1 and R2. For Aim 1, an interactive approach using tools of molecular biology to experimentally analyze structure-function relationship of TRH-Rs will be combined with computer simulations and chemical synthesis of novel TRH analogs. For Aim 2, an approach using molecular biology and biochemical and cell biological assays will be used. In Aim 3, two approaches, the yeast two-hybrid system and expression cloning, will be used to identify effector proteins that interact with the C-TERM tails of TRH-Rs.